Mechanisms of Heme Transport

描述（由申请人提供）：血液结合素（HPX）是对抗溶血状态下血红素介导的细胞损伤的第一道防线。它也是哺乳动物细胞中血红素转运的生理相关系统。广泛的长期目标是了解HPX摄取血红素的机制;确定HPXR在血红素摄取中的作用;确定血红素转运蛋白是否与HPXR密切相关;确定HPXR触发的信号何时以及如何导致基因调控，特别是保护性蛋白HO-1和MT-1的调控;并确定铜是否在HPX系统中的血红素摄取和信号传导中起作用;如果是，则确定所涉及的铜蛋白。具体目标是：1）。目的：研究55 Fe-血红素-HPX高亲和力、特异性转运血红素的过程。我们将评估的相对贡献的内吞作用，血红素分子的结构特征和减少血红素在血红素HPX复合物在细胞表面的血红素运输。为了实现血红素加氧酶-1调节所需的血红素摄取过程需要铜的目标，我们将确定铜螯合剂浴铜灵二磺酸盐是否抑制血红素摄取2）。使用设计的四种策略之一来克隆HPXR和3）。研究在HPXR的内吞作用之前，HPXR是否激活了由配体结合触发的细胞内信号级联。钴-原卟啉IX（CoPP）-HPX是HPXR配体，其激活信号级联而不需要四吡咯转运。我们将使用生物化学和形态学技术来确定CoPP-HPX是否停留在细胞表面或像血红素-HPX一样经历内吞作用。将在野生型和内吞缺陷型细胞中比较HPXR引发的调节结果。 这项研究的意义在于，它将提供有关血红素摄取的生物化学以及HPXR激活信号级联和基因调控（例如伴随血红素摄取的ho-1）的基本信息。这些在本质上是重要的，但也因为它们有助于我们理解血红素相关疾病。这些疾病包括溶血、创伤和缺血再灌注损伤，以及神经退行性疾病，如中风。氧化还原活性，营养金属在信号和基因调控中的新作用也可能是结果。

DESCRIPTION (provided by applicant): Hemopexin (HPX) is the first line of defense against heme-mediated damage to cells in hemolytic states. It is also a physiologically-relevant system of heme transport in mammalian cells. The broad long term objectives are to understand the mechanism of heme uptake by HPX; to define the role of the HPXR(s) in heme uptake; to establish whether a heme transporter is closely associated with the HPXR; to determine when and how signals that are triggered by the HPXR lead to gene regulation, particularly that of the protective proteins HO-1 and MT-1; and to establish whether copper plays in heme uptake and signaling in the HPX system; and, if so, to define the copper-protein(s) involved. The specific aims are: 1). To characterize the process of high affinity, specific heme transport from 55Fe-heme-HPX. We will assess the relative contributions of endocytosis, structural features of the heme molecule and reduction of heme in heme-HPX complexes at the cell surface for heme transport. Toward the goal that copper is required for the process of heme uptake needed for heme oxygenase-1 regulation, we will determine whether the copper chelator bathocuproine disulfonate inhibits heme uptake 2). To clone the HPXR using one of four strategies devised and 3). To investigate whether activation of intracellular signaling cascades by the HPXR, triggered by ligand binding, occurs before endocytosis of the HPXR. Cobalt-protoporphyrin IX(CoPP)-HPX is a HPXR ligand that activates signaling cascades without tetrapyrrole transport. We will use biochemical and morphological techniques to determine whether CoPP-HPX stays at the cell surface or undergoes endocytosis like heme-HPX. The regulatory consequences triggered by the HPXR will be compared in wild type and endocytosis-defective cells. The significance of this research is that it will provide fundamental information on the biochemistry of heme uptake as well as activation of signaling cascades and gene regulation (e.g. of ho-1 that accompanies heme uptake) by the HPXR. These are important intrinsically but also because they aid our understanding of heme-related diseases. These range from hemolysis, trauma and ischemia reperfusion injury but also neuro-degenerative conditions, such as stroke. Novel roles for redox-active, nutrient metals in signaling and gene regulation are also likely outcomes.